BPS2026 – SMLM and image contrast enhancement with BODIPY dye conjugates

Single molecule localization microscopy (SMLM) has revolutionized biological imaging by overcoming the diffraction limit (∼200 nm) through the temporal separation of closely spaced fluorophores, which requires controllable, sparse single-molecule blinking. We have reported that BODIPY (FL), a widely used conventional lipophilic dye (λ em ≈ 510 nm), can exhibit red-shifted single-molecule fluorescence (λ em ≈ 620 nm) via spontaneous ground state dimer (D II ) formation and can be leveraged for SMLM. Here, we demonstrate that the D II fluorescence yields enhanced image contrast due to the nonlinear relationship between local D II fluorescence and monomer concentration. We further expand this novel D II modality across a variety of existing BODIPY conjugates to image different organelles (ER, mitochondria, and Golgi) in live cells with enhanced contrast and super-resolution. We also find that D II formation is facilitated by lipid membrane interfaces but suppressed in other, less ordered environments, such as lipid droplet cores, suggesting the potential capability of environment-selective two-color staining with a single dye. To investigate this observation in a well-controlled model system, we have quantified the BODIPY fluorescence dependence on the dye orientation and lipid compositions using cell-sized liposomes. Together, our results establish the ground state dimerization of the extensive library of BODIPY conjugates as a simple strategy for SMLM and image contrast enhancement.